Microparticle organization

ABSTRACT

Methods and compositions are described for organizing nanoparticles or microparticles into nanostructures or microstructures using collagen as a template.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/265,729 filed Jun. 4, 2012 which is a U.S. National Stage applicationof PCT/US10/31922 filed Apr. 21, 2010 which claims priority to U.S.Provisional Application No. 61/171,237, filed Apr. 21, 2009, thecontents of which are hereby incorporated in their entirety herein.

FIELD OF THE INVENTION

The invention relates to microparticle organization for the preparationof organic and inorganic materials.

BACKGROUND OF THE INVENTION

Carbon nanotubes (CNTs) are hexagonal networks of carbon atoms formingseamless tubes, and each end can be capped with half of a fullerenemolecule. They were first reported in 1991 by Sumio Iijima who producedmulti-layer concentric tubes or multi-walled CNTs by evaporating carbonin an arc discharge. CNTs possess certain electronic and mechanicalproperties, making them candidates for applications relating tocomposite materials, nanoelectronics, sensors, and electron fieldemitters. CNTs can be utilized individually or as an ensemble to build avariety of devices. For instance, individual nanotubes have been used astips for scanning probe microscopy and as mechanical nano-tweezers.Ensembles of nanotubes have been used for field emission basedflat-panel displays, and bulk quantities of nanotubes may be used as ahigh-capacity hydrogen storage media. The electronic behavior of CNTs isclosely related to their structure, i.e., tip curvature, radius andcomposition, nanotube length, and chirality. Thus, there is a need formethods of arranging CNT structural elements, such as for electronicapplications, including the development of field emission devices(FEDs). However, there is no existing method capable of organizingnanotubes, especially over large scales.

SUMMARY OF THE DISCLOSURE

The disclosure is based, at least in part, on the discovery thatcollagen can be used to organize carbon nanotubes. Accordingly, in oneaspect, the invention features a method of organizing nanoparticles intoa nanostructure, comprising: contacting a collagen template with asolution comprising (i) collagen monomers in liquid crystalline phaseand (ii) nanoparticles; and assembling the collagen monomers into anordered collagen structure by neutralizing the solution in contact withthe collagen template, the ordered collagen structure directing theorganization of the nanoparticles into a nanostructure. In someembodiments, the solution is a still solution.

In some embodiments, the nanoparticle has a diameter of about 1 nm toabout 1 μm, about 10 nm to about 500 nm, about 20 nm to about 250 nm,about 30 nm to about 200 nm, about 1 nm to about 10 nm, about 10 nm toabout 20 nm, about 20 nm to about 30 nm, about 30 nm to about 40 nm, orabout 40 nm to about 50 nm. In other embodiments, the nanoparticle hasan aspect ratio of at least about 2, at least about 3, at least about 4,at least about 5, at least about 6, at least about 7, at least about 8,at least about 9, at least about 10, or more.

In some embodiments, the method further comprises contacting thenanoparticles with a crosslinking agent. In some embodiments, thecrosslinking agent is formaldehyde, hexamethylene diisocyanate,glutaraldehyde, a polyepoxy compound, gamma irradiation, ultravioletirradiation with riboflavin, transglutaminase, acyl azidesglycidylethers, diisocyanates, hexamethylenediisocyanate, bis-epoxide,carbodiimide, dimethylsuberimidate, nordihydroguaiaretic acid, lysyloxidase, or a combination thereof.

In other embodiments, the method further comprises removing the orderedcollagen structure from the nanostructure. In certain embodiments,removing the organized collagen structure comprises contacting theordered collagen structure with collagenase.

In some embodiments, the nanostructure is a single-walled carbonnanotube. In other embodiments, the nanostructure is a multi-walledcarbon nanotube.

In some embodiments, the still solution comprises about 1% to about 99%collagen monomers. In other embodiments, the still solution comprisesabout 1% to about 99% nanoparticles. In yet other embodiments, thesolution comprises a ratio of collagen monomers to nanoparticles ofabout 1% to about 99%.

In some embodiments, the collagen monomers comprise a nematic phase. Inother embodiments, the collagen monomers comprise a smectic phase. Inyet other embodiments, the collagen monomers comprise a cholestericphase.

In certain embodiments, the solution comprises about 30 mg/ml to about1000 mg/ml collagen monomers, about 30 mg/ml to about 500 mg/ml, about40 mg/ml to about 400 mg/ml, about 50 mg/ml to about 300 mg/ml, about 60mg/ml to about 200 mg/ml, about 70 mg/ml to about 150 mg/ml, about 80mg/ml to about 125 mg/ml, about 90 mg/ml to about 120 mg/ml, or about100 mg/ml collagen monomers.

In some embodiments, the method includes neutralizing the solution byadjusting the solution to a pH of about 5 to about 10, about 5.5 toabout 9.5, about 6 to about 9, about 6.5 to about 8.5, or about 6.5 toabout 8.

In other embodiments, the method includes neutralizing the solution incontact with the template at about 10° C. to about 39° C., at about 10°C. to about 35° C., about 15° C. to about 30° C., or about 20° C. toabout 25° C.

In certain embodiments, the method further comprises applying anelectric charge to the collagen template.

In particular embodiments, the collagen template comprises one or moreguidance structures. In specific embodiments, the one or more guidancestructures are one or more internal guidance structures and the collagentemplate is placed in a stationary position within the solution. In someembodiments, the guidance structures comprise a surface having a patternof hydrophobic and hydrophilic stripes.

In other embodiments, the one or more internal guidance structurescomprise a high aspect ratio geometry. In particular embodiments, theone or more internal guidance structures comprise a minor length scaleof between about 14 nm and about 20 μm, for example, between about 20 nmand about 15 μm, between about 25 nm and about 10 μm, between about 30nm and about 5 μm, between about 40 nm and about 100 nm, between about50 nm and about 90 nm, between about 60 nm and about 80 nm, or about 70nm.

In some embodiments, one or more of the internal guidance structurescomprise a biodegradable material. In certain embodiments, thebiodegradable material is silk, PLGA, or a PLA-type material (such asPDLA, PLLA, or PDLLA).

In yet other embodiments, the collagen template comprises a plurality ofexternal guidance structures. In some embodiments, the external guidancestructures have an interstructure distance of about 2 μm to about 200μm, for example about 4 μm to about 175 μm, about 8 μm to about 150 μm,about 10 μm to about 125 μm, about 20 μm to about 100 μm, about 30 μm toabout 90 μm, about 40 μm to about 80 μm, or about 50 μm to about 70 μm.In some embodiments, the collagen template comprises one or moreinternal guidance structures and one or more external guidancestructures.

In certain embodiments, the collagen template comprises a cylindricaltube, two concentric cylindrical tubes, or two concentric hemispheres.In some embodiments, the collagen template comprises a cylindrical tubehaving an inner diameter of about 100 μm to about 1 mm, for exampleabout 125 μm to about 900 μm, about 150 μm to about 800 μm, about 175 μmto about 700 μm, about 200 μm to about 600 μm, about 300 μm to about 500μm, or about 400 μm to about 450 μm. In other embodiments, the collagentemplate comprises two concentric cylinders with a gap width of about 2μm to about 4 mm, for example, about 4 μm to about 2 mm, about 8 μm toabout 1 mm, about 10 μm to about 900 μm, about 20 μm to about 800 μm,about 30 μm to about 700 μm, about 40 μm to about 600 μm, about 50 μm toabout 500 μm, about 100 μm to about 400 μm, or about 200 μm to about 300μm. In yet other embodiments, the collagen template comprises twoconcentric hemispheres with a gap width of about 2 μm to about 4 mm, forexample, about 4 μm to about 2 mm, about 8 μm to about 1 mm, about 10 μmto about 900 μm, about 20 μm to about 800 μm, about 30 μm to about 700μm, about 40 μm to about 600 μm, about 50 μm to about 500 μm, about 100μm to about 400 μm, or about 200 μm to about 300 μm.

In some embodiments, the collagen template comprises a scaffold thatmimics a cornea, a ligament, a tendon, a meniscus, an intervertebraldisk, or articular cartilage.

In certain embodiments, the collagen monomers are selected from thegroup consisting of Type I collagen monomers, Type II collagen monomers,Type III collagen monomers, Type V collagen monomers, Type XI collagenmonomers, an MMP-resistant mutant thereof, and combinations thereof Inother embodiments, the collagen monomers are selected from the groupconsisting of atelo-collagen monomers, tropocollagen monomers,procollagen monomers, and combinations thereof.

In particular embodiments, the solution comprises a buffer or saltselected from the group of CaCl₂, NaOH, NaCl, Na₂HPO₄, NaHCO₃, Hepes,PBS, Trizma base, Tris-HCl, cell culture media, and combinationsthereof.

In yet other embodiments, the solution comprises one or moreco-nonsolvency agents. In certain embodiments, the co-nonsolvency agentis polyethylene glycol, hyaluronic acid, a glycosaminoglycan, aproteoglycan, or a combination thereof In some embodiments, theglycosaminoglycan is chondroitin sulfate, hyaluronic acid, heparin,heparin sulfate, keratin sulfate, or dermatan sulfate.

In other embodiments, the solution further comprises a collagen bindingagent. In some embodiments, the collagen binding agent is aproteoglycan, a glycoprotein, a collagen-binding portion thereof, or acombination thereof In certain embodiments, the proteoglycan is lμmican,decorin, biglycan, perlecan, versican, fibromodulin, aggrecan, sydecanor a combination thereof In other embodiments, the glycoprotein isfibronectin, laminin, osteonectin, or a combination thereof.

In yet other embodiments, the ordered collagen structure is about 100 μmto about 30 cm in length, for example, about 200 μm to about 20 cm,about 400 μm to about 10 cm, about 500 μm to about 5 cm, about 750 μm toabout 1 cm, about 1 mm to about 500 mm, about 10 mm to about 400 mm,about 50 mm to about 300 mm, about 100 mm to about 200 mm, or about 100mm to about 150 mm.

In certain embodiments, the method further comprises contacting thecollagen monomers in the ordered collagen structure with a crosslinkingagent. In some embodiments, the crosslinking agent is formaldehyde,hexamethylene diisocyanate, glutaraldehyde, a polyepoxy compound, gammairradiation, ultraviolet irradiation with riboflavin, transglutaminase,acyl azidesglycidyl ethers, diisocyanates, hexamethylenediisocyanate,bis-epoxide, carbodiimide, dimethylsuberimidate, nordihydroguaiareticacid, lysyl oxidase, or a combination thereof.

In other embodiments, the method further comprises modulating thesurface energy of the guidance structures. In some embodiments, thesurface energy is modulated by plasma cleaning, silanization, orhydrophobic/hydrophilic bonding.

In other embodiments, the nanoparticle is a nanoparticle describedherein.

The following figures are presented for the purpose of illustrationonly, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representation of a crossed polarized microscopy image ofcollagen nanotube constructs showing the relatively aligned arrays.

FIG. 1B is a representation of a crossed polarized microscopy image ofcollagen nanotube constructs showing the relatively aligned arrays.

FIG. 1C is a representation of a polarized microscopy image of collagennanotube constructs taken at the interface between two layers within aconstruct showing orthogonal lamellae.

FIG. 2A is a representation of an en face scanning electron microscopy(SEM) image of collagen nanotube constructs at low magnification showingthat the construct comprises aligned arrays.

FIG. 2B is a representation of a cross sectional SEM image of collagennanotube constructs at high magnification showing multi-layerconstructs.

FIG. 2C is a representation of a cross sectional SEM image of collagennanotube constructs at low magnification.

FIG. 2D is a representation of a cross sectional SEM image of collagennanotube constructs at high magnification showing multi-layerconstructs.

FIG. 3 is a representation of an SEM image of 100% carbon nanotubesconcentrated against 40% PEG for 2 weeks. These appear conductive, butnot completely organized.

FIG. 4A is a schematic illustration of a method of designing andfabricating lamellar structures.

FIG. 4B is a schematic illustration of methods of producingcollagen-steerable microparticles.

FIG. 5 is a schematic illustration of a method of particle arrangementby collagen.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein,including GenBank database sequences, are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Definitions

As used herein, a “collagen template” is a three-dimensional structureor substrate that controls collagen fibril organization to produce anordered collagen structure. A collagen template creates a zone of localinfluence within a solution of collagen. A collagen template comprisesone or a plurality of guidance structures. An ordered collagen structurecan be used to organize nanoparticles or microparticles as describedherein.

As used herein, a “guidance structure” is a structure with a high aspectratio with a minor length scale of between about 14 nm and about 20 μm.The guidance structure is defined by the operative length scale of acollagen monomer.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 20%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

As used herein, “interstructure distance” means the distance between theouter surfaces of two adjacent guidance structures.

As used herein, “alignment” in reference to collagen fibrils means thatmost of the fibrils in the same radial plane in a tube wall run roughlyparallel to each other. It is not meant that every fibril must beparallel to every other fibril in the plane, but that a generalalignment pattern must be discernable.

As used herein, a “fibril” is an association of several collagenmonomers into a structure that appears fibrous with suitablemagnification.

As used herein, “collagen” means a protein component of an extracellularmatrix having a tertiary structure that includes polypeptide chainsintertwining to form a collagen triple helix or having a characteristicamino acid composition comprising Gly-X-Y repeat units, or a fragmentthereof Collagens can be any collagen known in the art (e.g., one ofcollagen Type 1-29).

As used herein, “nanoparticle” means any particle having a minordiameter of about 1 nm to about 1,000 nm and having an aspect ratio ofat least about 2.

As used herein, “microparticle” means any particle having a minordiameter of about 1 μm to about 1,000 μm and having an aspect ratio ofat least about 2.

As used herein, “carbon nanotube” refers to a hollow cylindrical articlecomposed primarily of carbon atoms. A carbon nanotube can have adiameter of about 1 nm to about 1 μm and a ratio of length to diameter(i.e., aspect ratio) of at least about 2.

General

The methods described herein are based, at least in part, on thediscovery that microparticles or nanoparticles, such as carbonnanotubes, can be organized into microstructures or nanostructures usingcollagen as a template. Producing aligned, single-walled carbonnanotubes (swCNTs) has numerous potential applications from thegeneration of transistors and electrical switches to the production ofexceedingly strong materials. This disclosure uses the ability ofcollagen monomers to form organized aligned structures when highlyconcentrated (as described in, e.g., PCT/US09/40364) to provide aguidance nanotube template for co-dispersed carbon nanotubes. Thisapproach is feasible because single-walled carbon nanotubes aregeometrically similar to collagen monomers (300 nm×1.5 nm). Thisdisclosure includes the ability to produce organized swCNTs bydispersing the swCNTs (1%-75% molar fraction relative to collagen) in adense solution of collagen monomers (30 mg/ml-500 mg/ml). Collagen,which behaves as a liquid crystal, strongly influences the organizationof the swCNTs because they are geometrically similar. Following theproduction of organization in the mixed solution of collagen and swCNTs,the collagen can be removed by a number of methods. The organization ofthe swCNTs during collagen removal can be preserved, such as bycrosslinking functional groups or by maintaining a high osmoticpressurization to confine the swCNTs. Collagen can be removed in anumber of ways, such as enzymatically by exposure to collagenase(bacterial, MMP and others), or by heat denaturation followed byexposure to gelatinase leaving behind the organized swCNTs provided theyare sufficiently stabilized.

Microparticles and Nanoparticles

The methods described herein can be used to organize microparticles andnanoparticles into microstructures or nanostructures. In someembodiments, the nanostructures are carbon nanotubes. Carbon nanotubesare carbon nanostructures in the form of tubes, ranging in general indiameter from about 0.5-200 nm, (more typically for single-walled carbonnanotubes from about 0.5-5 nm). The aspect ratio of nanotube length tonanotube diameter is greater than about 5, ranges from 10-2000 and moretypically 10- 100. Carbon nanotubes may be single-walled nanotubes (asingle tube) or multi-walled comprising with one or more smallerdiameter tubes within larger diameter tubes. Carbon nanotubes areavailable from various sources, including commercial sources (e.g.,Helix Materials Solutions, Richardson, Tex.), or synthesis employing,among others, arc discharge, laser vaporization, the high pressurecarbon monoxide processes.

Exemplary methods for synthesis of carbon nanotubes are described in,e.g., U.S. Pat. No. 6,183,714; WO 00/26138; WO 03/084869; WO 02/16257;Thess et al., Science 273:483 (1996); Journet et al., Nature 388, 756(1997); Nikolaev et al., Chem. Phys. Lett. 313:91 (1999); Kong et al.,Chem. Phys. Lett. 292: 567 (1998); Kong et al., Nature 395:878 (1998);Cassell et al., J. Phys. Chem. 103:6484 (1999); Dai et al., J. Phys.Chem. 103:11246 (1999); and Li et al., Chem. Mater. 13:1008 (2001).

A method for separating single-walled carbon nanotubes by diameter andconformation based on electronic and optical properties has beenreported (WO 03/084869). The method can be used to prepare carbonnanotube preparations having enhanced amounts of certain single walledcarbon nanotube types (see, e.g., Zheng et al., Science 302:1545(2003)).

Other microparticles and nanoparticles include organic or inorganicmaterials. In particular instances, the microparticles or nanoparticlesare ceramics, metals, or composites. Nonlimiting examples are metallic(e.g., Ni, Pt, Au), semiconducting (e.g., Si, InP, GaN), and insulating(e.g., SiO₂, TiO₂) materials.

Collagen

The methods described herein involve using collagen to produce organizedmicrostructures or nanostructures. Collagen is the most abundant proteinin the extracellular matrix (ECM) of vertebrates and is the most commonstructural molecule in tensile load-bearing applications.

More than 29 different collagenous sequences are known. Fibrillarcollagens (e.g., Types I, II, III, V and XI) are the principalstructural component in load-bearing extracellular matrix (ECM), whichprovides a network for cells to interact and form three dimensional,multi-cellular organisms. Collagen possesses a linear-helical structurecomprising three left-handed helical alpha chains whose complementaryamino acid sequence results in the formation of a right-handedsupramolecular triple helix. Collagen contains the repetitive sequenceamino acid sequence Gly-X-Y, where×and Y are usually proline andhydroxyproline, respectively.

As described herein, collagen is not a passively manipulated element,but rather a principal component in a cooperative engineering materialsystem, a system that significantly enhances the ability of fibroblasticcells to produce and optimize load-bearing tissue.

Any known collagen can be used in the methods described herein and canbe isolated or derived from a natural source, manufactured biochemicallyor synthetically, produced through genetic engineering, or producedthrough any other means or combinations thereof. In addition, collagenis commercially available (e.g., from Inamed Biomaterials, Fremont,Calif.; and FibroGen, Inc., San Francisco, Calif.). Natural sourcesinclude, but are not limited to, collagens produced by or containedwithin the tissue of living organisms (e.g., cows, pigs, birds, fish,rabbits, sheep, mice, rats, and humans). Further, natural collagen canbe obtained from, for example, tendons, bones, cartilage, skin, or anyother organ by any known extraction method. Exemplary sources includerat tail tendon and calf skin.

Some collagens that are useful in the methods described herein include,but are not limited to, collagen Types I, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX. Syntheticcollagen can include collagen produced by any artificial means, andnumerous methods for producing collagens and other proteins known in theart can be used. For example, synthetic collagen can be prepared usingspecific sequences, such as specific amino acids that are the same orthat differ from natural collagen. Engineered collagen can be producedby any method known in the art including, for example, polypeptidesynthesis.

Other Molecules

In addition to collagen, other molecules can be used to organizemicroparticles or nanoparticles into microstructures or nanostructuresin the methods described herein. Such molecules include molecules withself-organizing and/or liquid crystalline properties. Specific,nonlimiting examples include actin and microtubules.

Methods of Organizing Microstructures and Nanostructures

Concentrating and Precipitating Collagen

In some instances, the methods described herein include confining asolution of collagen monomers within a collagen template having adefined confinement geometry (e.g., having defined external guidancestructures). The solution can include any type of collagen monomers.

The collagen in solution can be in a liquid crystalline phase, e.g., innematic, smectic, or cholesteric phase. In certain instances, theconcentration of collagen monomers in the solution is between about 30mg/ml and about 500 mg/ml. In other instances, the collagen solutionincludes a buffer. Examples of buffers include, without limitation,CaCl₂, NaOH, NaCl, Na₂HPO₄, NaHCO₃, Hepes, PBS, Tris, cell culturemedia, and combinations thereof

By confining the solution of collagen monomers within external guidancestructures, the collagen monomers are induced to precipitate and to formcollagen arrays having a desired architecture. In one exemplary method,collagen monomers are concentrated to about 100 mg/ml and are confinedbetween featureless planar glass coverslips separated by about 40μ,leading to fibril precipitation from the solution with a high-degree ofalignment in planes parallel to the coverslips. In this situation, thecoverslips provide external guidance structures that induce thealignment of the fibrils. Further, the collagen fibrils can form layersin which the orientation of the alignment of the fibrils can changedirection, forming a natural load-bearing structure similar to nativecollagen organization found in cornea, bone, blood vessel intima oradventitia, and annulus fibrosus. Thus, the concentration andconfinement of collagen in axially symmetric geometries can result inthe formation of structures similar to any collagenous tissue, such asligament or tendon.

In other methods, the local organization of collagen is controlled byusing internal collagen templates (e.g., internal guidance structures).While not wishing to be bound by theory, it is believed that suchinternal collagen templates mimic embedded fibroblasts (or fibroblastfilipodia) to influence the local organization of collagen fibrils. Incertain instances, the internal collagen template is made of abiodegradable polymer. Nonlimiting examples of biodegradable polymersinclude silk, poly(lactide), poly(glycolic acid),poly(lactide-co-glycolide), poly(caprolactone), polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates, and degradable polyurethanes.

In one exemplary method, an internal collagen template is made of finedegradable filaments that are woven into a sparse scaffold. The scaffoldcan then be immersed in a solution of concentrated collagen monomers,which induces the alignment of the precipitating collagen to follow theinternal collagen template. The spacing and size of the internalcollagen template (e.g., biodegradable filaments) can be arranged toresult in a particular collagen fibrillar organization of interest.Thus, the use of internal guidance structures allows internal controlover the organization of collagen, such as solutions of collagenmonomers confined within a particular geometry (e.g., within externalguidance structures).

Organizing Microstructures and Nanostructures

As described herein, the concentrated and precipitated collagen isorganized into ordered collagen structures, which organizemicrostructures and nanostructures, such as carbon nanotubes. Bycodispersing microstructures or nanostructures with a solution ofcollagen monomers followed by inducing the alignment of collagenmonomers into collagen fibrils, the collagen fibrils form an organizedcollagen structure that directs the organization of the microstructureor nanostructure, such as a carbon nanotube. The ratio of collagenmonomers and microstructures or nanostructures in solution can vary. Inparticular examples, the ratio of collagen monomers to microstructuresor nanostructures is about 1% to about 99%.

Modifying External and Internal Templates

The external and internal collagen templates (e.g., external andinternal guidance structures) can be modified to influence the collagenfibrillar organization. For example, using known methods, the surfacesof the external and internal collagen templates can be plasma cleaned,patterned, or functionalized in other ways to control the localorganization of the interfacing fibrils to produce collagen arrays. Inparticular methods, the surfaces of the external and/or internalcollagen templates are silanated or carbodiimidated using known methods.

In some instances, the surface charges of collagen molecules can be usedto direct the process of collagen assembly by applying an electriccharge to one or more surfaces of the external and/or internal collagentemplates. In particular methods, collagen molecules can be confinedbetween two metallic plates containing an electrical field to direct theassembly of collagen. In other situations, the amount of free ioncharges in the solution can be altered to change the degree of variationin alignment between layers.

Auxiliary Molecules

The methods described herein can include the use of one or moreauxiliary molecules, e.g., collagen modulating molecules such asextracellular matrix molecules. Such molecules include, but are notlimited to, proteoglycans (such as perlecan, versican, syndecan,decorin, lμmican, and biglycan), proteoglycan core proteins,glycosaminoglycans (such as hyaluronic acid, chondroitin-4 sulfate,chondroitin-6 sulfate, dermatan sulfate, heparin, heparin sulfate, andkeratan sulfate), Type V collagen, fibronectin, or any molecule thatcompetes with collagen for available water (such as polyethyleneglycol). Such molecules can be added to a solution containing collagenmonomers prior to or following the precipitation of collagen asdescribed herein.

These auxiliary molecules can be used to increase the rate of theprocess and higher order organization. The ratio of auxiliary moleculeto collagen can depend on the type of the molecules, and can range fromabout 10% to about 50%.

Methods of Strain Stabilization, Monomer incorporation and EnzymaticDegradation of Collagen

Some of the methods described herein are based, at least in part, on thediscovery of a strain-dependent mechanism that can modulate collagenfibril susceptibility to enzymatic degradation. This mechanism canproduce a physicochemical change at the matrix level that is bound tofibril strain. Based on this “strain-stabilization of collagen”mechanism, tensile strains can provide a robust signal, leading to aload-controlled differential degradation (catabolism) of collagen inextracellular matrix. Further, tensile strains on collagen fibrils canprovide a signal, leading to the incorporation of collagen monomers intoloaded fibrils (this is monomer incorporation). In some instances, theadaptive remodelling response of load-bearing ECM can be controlled bycollagen and its complement enzymes (e.g., bacterial collagenase, MMPs,and cathepsins), which couple the control signal (i.e., mechanical load)to a physicochemical change in the collagen molecules or fibrils. Inaddition, this mechanism can relieve fibroblasts of the burden of“knowing” which fibrils to degrade during remodelling and which toreinforce. Based on this mechanism, load-stimulated fibroblasts canproduce a load-adapted morphological change during, e.g., epigeneticconnective tissue remodelling, repair, homeostasis and disease.

In some instances, collagen is precipitated as described herein, and thecollagen organization is further refined by subjecting the initialcollagenous construct to cross-linking, mechanical strain, and/orenzymes to cull unwanted (unstrained) fibrils (see, e.g., Ruberti etal., Biochem. Biophys. Res. Commun. 336:483-489 (2005)).

Mechanical strain can be applied to collagen fibrils using, e.g., amicrochamber (see, e.g., PCT/US09/40364). In such methods, the collagencan be fixed to grips in a microchamber by, e.g., direct clamping or byadhesives (such as cyanoacrylates). In some situations, the collagen isaffixed to functionalized micropipettes as described herein. During theloading of mechanical strain, auxiliary molecules can optionally beincluded. In addition, hydroxyapetite and noncollagenous proteins can beadded to calcify the system during loading.

In some situations, prior to the loading of the construct, collagenfibrils or carbon nanotubes are cross-linked, such as to increasestability. Any suitable crosslinking agent known in the art can be usedincluding, without limitation, formaldehyde, hexamethylene diisocyanate,glutaraldehyde, polyepoxy compounds, gamma irradiation, and ultravioletirradiation with riboflavin. The crosslinking can be performed by anyknown method (see, e.g., Bailey et al., Radiat. Res. 22:606-621 (1964);Housley et al., Biochem. Biophys. Res. Commun. 67:824-830 (1975);Siegel, Proc. Nati. Acad. Sci. U. S. A. 71:4826-4830 (1974); Mechanic etal., Biochem. Biophys. Res. Commun. 45:644-653 (1971); Mechanic et al.,Biochem. Biophys. Res. Commun. 41:1597-1604 (1970); and Shoshan et al.,Biochim. Biophys. Acta 154:261-263 (1968)).

Enzymes

The methods described herein can include degradation of organizedcollagen structures after microstructure or nanostructure organization.Degradation can be performed using enzymes, such as collagen-degradingenzymes including, without limitation, collagenase (e.g., bacterialcollagenase), cathepsin, and matrix metalloproteases (MMPs). Suchenzymes are commercially available, e.g., from Sigma Aldrich. The enzymecan be added to the culture mediμm which is then introduced to thecollagen or other structural material.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 In Vitro Organization of Carbon Nanotubes usingCollagen

Carbon nanotubes were mixed with collagen solution (3 mg/mL acidicsolution of pepsin-extracted, atelo, type I bovine collagen monomers(PURECOL®, Inamed, Freemont, Calif.)) in the ratio of 1:1. Theconcentration procedure was performed by dialyzing the mixture against40% solution of polyethylene glycol (PEG, 20 kMWCO, Sigma-Aldrich, St.Louis, Mo.) at 4° C. The dialyzing procedure proceeded until theconcentration of the collagen molecules reached the range of 175±25mg/mL. Then to reach the final concentration of 375±25 mg/mL, thedialyzing was further proceeded by injecting the solution into adialysis cassette and dispensing the cassette in the PEG solution. Thecollagen solution was then neutralized (by titrating the PEG solutionand letting the system to equilibrate) and transferred to a 37° C. oven.The SWNT/collagenous constructs were then carefully removed for furtherultra-structural assessments using Differential interference contrast(DIC), Scanning Electron Microscopy (SEM), and standard TransmissionElectron Microscopy (sTEM). At least seven experiments at eachconcentration range were performed.

As shown in FIGS. 1-3, there was very little disruptive influence of thenanotubes on the collagen organization at low loading concentrations ofnanotubes into the liquid crystal collagen. At higher concentrations ofnanotubes there was less organization.

Example 2 Collagen-Mediated Organization of Microstructures

The ability of collagen to organize microstructures is assessed by firstprocessing coatings with lamellar defects and producing microparticlesthat can be combined with collagen for alignment into organizedmicroparticles.

Systematic Processing and Post-Processing of Ceramic Coatings withLamellar Defects

Specific process input values are determined to fabricate lamellarstructures (LS) with intended plate thickness, width and gap spacingdistribution. First, ceramic powders (such as YSZ, Al₂O₃, or TiO₂) areobtained from commercial sources. Powders are fed into Air Plasma Spray(APS—high temperature, low velocity) and High Velocity Oxy Fuel(HVOF—lower temperature, high velocity) torches (Center for ThermalSpray Research, SUNY Stony Brook). Torch input parameters (current, gasflow rate) are correlated with in-flight particle state (Temperature,Velocity) to calculate ‘melting index’ (MI). In addition, the effects ofinput parameters on interfacial geometry (thickness, morphology,orientation, connectivity) within the lamellar structure are assessed.Low-strain in-plane mechanical cycling is used as a post-processingtechnique for further control over gap geometry, and enhanced collageninfiltration. Coatings are deposited on constant strain cantilevers, andmechanically cycled via beam bending to produce the desired strains.

For assessment, LS architecture is examined via optical microscopy andSEM in cross-section, using known image analysis methods. In particular,interface morphology (roughness) is examined locally using HRSEM. Inaddition, supplemental information is obtained via small angle neutronscattering (SANS).

This method provides specific design space of inorganic lamellarstructures, for (i) infiltration with collagen or other organics and(ii) realizable architectures for input into multi-scale computationalmodels. LS are designed with ‘brick’ particle thickness controllablewithin approximately 250 nm, gap spacing within approximately 10 nm, andgap length within half a ‘brick’ particle width. Optimum conditions forcollagen infiltration and LS regularity are developed via a two stageprocess of (i) high velocity, high-flattening impact of particles (withsmall interfacial gaps) and (ii) increase in gap area via post-depositfatigue processing.

Systematic Processing of Ceramic Particles with Designed Geometry

A high-volume process is established for production of impactedparticles with highly controllable geometry, by recourse to TS impactprocessing. Powders (down to 10 micron diameter) and torch parametersare selected as described in Example 2 and are used to create andcollect individual particles that are not bonded to the substrate noreach other. A novel ‘waterfall processing’ system is used, which is asubstrate with a thin downward flow of liquid (such as water) leading toa collection unit. Molten particles impact on the substrate and flatten,but adhesion is prevented by the liquid layer and the particles aredriven downward for collection. Flattening ratio is designed via processparameters and fluid impact models known in the art. In addition,substrates are prepared with varying degrees of roughness or waviness,to produce particles having interlocking capabilities. Finally,particles are impacted on substrates at non-normal orientation (as shownin FIG. 4), and in combination with ‘channels’, to produce higher-aspectratio particles.

For assessment, particles are pulled out of the collection unit via‘scooping’ on a substrate, and examined under optical microscopy andSEM. Distributions of properties, such as aspect ratios, are correlatedwith batch conditions. In addition, some process conditions lead to a‘bubbly’ bottom surface of splats, due to outgassing of the liquidlayer, or impact-induced depressurization. In some cases, the bubbleshave sub-micron dimension, which lead to further design considerationsfor interaction with collagen, i.e. increased adhesion.

Organization of Microparticle “Bricks” by Collagen

As shown in FIG. 5, particles (depicted as “bricks”) are combined withcollagen in solution in the presence of external templates with orwithout internal templates. The assembly of collagen into organizedcollagen structures “drives” and pulls particles into organizedarrangements, as depicted.

Equivalents

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of organizing nanoparticles into a nanostructure,comprising: contacting a collagen template with a still solutioncomprising (i) collagen monomers in liquid crystalline phase and (ii)nanoparticles; and assembling the collagen monomers into an orderedcollagen structure by neutralizing the solution in contact with thecollagen template, the ordered collagen structure directing theorganization of the nanoparticles into a nanostructure.
 2. The method ofclaim 1, further comprising crosslinking functional groups on thenanoparticles to stabilize the nanostructure.
 3. The method of claim 1,further comprising removing the ordered collagen structure from thenanostructure.
 4. The method of claim 3, wherein removing the organizedcollagen structure comprises digestion with collagenase.
 5. The methodof claim 1, wherein the nanostructure is a single-walled carbonnanotube.
 6. The method of claim 1, wherein the nanostructure is amulti-walled carbon nanotube.
 7. The method of claim 1, wherein thestill solution comprises about 1% to about 99% collagen monomers.
 8. Themethod of claim 1, wherein the still solution comprises about 1% toabout 99% nanoparticles.
 9. The method of claim 1, wherein the solutioncomprises a ratio of collagen monomers to nanoparticles of about 1% toabout 99%.
 10. The method of claim 1, wherein the collagen monomerscomprise a nematic phase.
 11. The method of claim 1, wherein thecollagen monomers comprise a smectic phase.
 12. The method of claim 1,wherein the collagen monomers comprise a cholesteric phase.
 13. Themethod of claim 1, wherein the solution comprises about 30 mg/ml toabout 500 mg/ml collagen monomers.
 14. The method of claim 1, whereinthe neutralizing step comprises adjusting the solution to a pH of about5 to about
 10. 15. The method of claim 14, further comprisingneutralizing the solution in contact with the collagen template at about10° C. to about 39° C.
 16. The method of claim 1, wherein the collagentemplate comprises one or more guidance structures.
 17. The method ofclaim 16, wherein the one or more guidance structures are one or moreinternal guidance structures and the collagen template is placed in astationary position within the solution.
 18. The method of claim 17,wherein the one or more internal guidance structures comprise a highaspect ratio geometry.
 19. The method of claim 18, wherein the one ormore internal guidance structures comprise a minor length scale ofbetween about 14 nm and about 20 μm.
 20. The method of claim 17, whereinthe one or more internal guidance structures comprise a biodegradablematerial.
 21. The method of claim 1, wherein the collagen templatecomprises a plurality of external guidance structures having aninterstructure distance of about 2 μm to about 200 μm.
 22. The method ofclaim 1, wherein the collagen template comprises a cylindrical tube, twoconcentric cylindrical tubes, or two concentric hemispheres.
 23. Themethod of claim 22, wherein the collagen template comprises acylindrical tube having an inner diameter of about 100 μm to about 1 mm.24. The method of claim 22, wherein the collagen template comprises twoconcentric cylinders with a gap width of about 2 μm to about 4 mm. 25.The method of claim 22, wherein the collagen template comprises twoconcentric hemispheres with a gap width of about 2 μm to about 4 mm. 26.The method of claim 1, wherein the collagen template comprises one ormore internal guidance structures and one or more external guidancestructures.
 27. The method of claim 1, wherein the solution comprisesone or more co-nonsolvency agents.
 28. The method of claim 1, whereinthe solution further comprises a collagen binding agent.
 29. The methodof claim 1, further comprising applying an electric charge to thecontacted collagen template.
 30. The method of claim 1, wherein theordered collagen structure is about 100 μm to about 30 cm in length.